ATP-Dependent formation of phosphatidylserine-rich vesicles from the endoplasmic reticulum of leek cells

Abstract

Leek (Allium porrum) plasma membrane is enriched in phosphatidylserine (PS) by the vesicular pathway, in a way similar to that already observed in animal cells (B. Sturbois-Balcerzak, D.J. Morre, O. Loreau, J.P. Noel, P. Moreau, C. Cassagne [1995] Plant Physiol Biochem 33: 625-637). In this paper we document the formation of PS-rich small vesicles from leek endoplasmic reticulum (ER) membranes upon addition of ATP and other factors. The omission of ATP or its replacement by ATPgamma-S prevents vesicle formation. These vesicles correspond to small structures (70-80 nm) and their phospholipid composition, characterized by a PS enrichment, is compatible with a role in PS transport. Moreover, the PS enrichment over phosphatidylinositol in the ER-derived vesicles is the first example, to our knowledge, of phospholipid sorting from the ER to ER-derived vesicles in plant cells.

Figure 1

Confocal laser scanning micrographs of the Golgi apparatus and ER in leek root cells. Projection of optical sections from confocal data sets. Magnification, ×1400. Top, Immunolocalization of the ER stained with anti-HDEL. The ER appears as a fine network, often radiating out from the nucleus through the cell. There is often staining in the nuclear membrane as well. Bottom, Immunolocalization of the Golgi apparatus in leek root cells stained with JIM 84. Golgi stacks appear scattered throughout the cytoplasm.

Figure 2

Immunoblots of ER and Golgi fractions with anti-HDEL and JIM 84 antibodies. SDS-PAGE and immunostaining were done as explained in Methods. Lanes corresponding to the membranes of the ER and the Golgi-enriched membrane fraction are indicated, respectively, as ER and Golgi. M_rs (in thousands) of classic standards are also indicated. For each lane, 5 μg of proteins was loaded.

Figure 3

Electron micrograph of the ER-derived vesicles isolated on discontinuous Suc-density gradients. Incubation of ER membranes with or without ATP and isolation of ER-derived vesicles were performed as described in Methods. Membranes were fixed and prepared for electron microscopy, as described in Methods. A, ER-derived vesicles (TV[+]) obtained from the ER membranes incubated in the presence of ATP. B, The same fraction as in A but at a higher magnification. C, Membrane fraction (TV[−]) isolated from the ER membranes incubated in the absence of ATP. Bars = 1 μm.